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Invitrogen™ MAX Efficiency™ Stbl2™ Competent Cells

Catalog No. 10268019
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Catalog No. 10268019 Supplier Invitrogen™ Supplier No. 10268019
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Description

Includes

MAX Efficiency Stbl2 Competent Cells: 5 vials, 200μL each (total of 1mL), pUC19 DNA (0.01μg/mL): 1 vial, 100μL, SOC Medium: 1 bottle, 6mL

MAX Efficiency Stbl2 Competent Cells are high-efficiency chemically competent cells specifically designed for cloning unstable inserts.

MAX Efficiency Stbl2 Competent Cells are high-efficiency chemically competent cells specifically designed for cloning unstable inserts. In addition to recA1, a unique set of genetic markers allow for stable cloning of direct repeat and retroviral sequences and tandem array genes. The Stbl2 strain was used for the stable propagation of a plasmid containing approximately 100 direct repeats of 32 bp as well as full-length infectious simian immunodeficiency virus (SIV) clone can be maintained. This strain has also been demonstrated to stably propagate plant virus sequences such as potato virus Y (PVY).

MAX Efficiency Stbl2 Competent Cells are a derivative of the JM109/J5 strain. Competent cells are prepared by a patented modification of the procedure of Hanahan. The mcrA mutation and the mcrBC-hsdRMS-mrr deletion allow for the cloning of methylated genomic sequences.

MAX Efficiency Stbl2 Competent Cells offer:
• >1 x 109 transformants/μg plasmid DNA for cloning rare sequences
• Elimination of mcrBC-hsdRMS-mrr and mutation of mcrA that allows cloning of methylated genomic sequences
• A unique genotype that stabilizes direct repeats and retroviral sequences such as SIV and HIV
• Greatly increased plasmid yield and quality due to endA1 mutation
• recA1 for reduced occurrence of nonspecific recombination in cloned DNA

Genotype
F- mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 lon gyrA96 thi supE44 relA1 λ- Δ(lac-proAB)

Note: For optimal performance, expression in S.O.C. Medium and incubation on antibiotic plates at 30°C is recommended.

Find the strain and format that fits your needs
• A variety of Stbl strains are available with different genetic background or suitable for electroporation.
• The Stbl3 strain is available in MultiShot format for high throughput applications.

Order Info

Shipping Condition: Dry Ice

Specifications

Product Type Chemically Competent Cells
Contains F' Episome No
Improves Plasmid Quality Yes (endA1)
Cloning Methylated DNA Yes (mcrB, mrr)
Transformation Efficiency Level High Efficiency (>1 x 109 cfu/μg)
Content And Storage • MAX Efficiency Stbl2 Competent Cells (5 x 200 μL); store at –80°C
• pUC19 DNA (100 μL at 0.01 μg/mL); store at –20°C
• S.O.C. Medium (6 mL); store at 4°C or room temperature
Antibiotic Resistance Bacterial No
Cloning Unstable DNA Yes (recA1)
Blue/White Screening No
High-throughput Compatibility Low
Plasmid May be used for plasmids >20 kb
Preparing Unmethylated DNA No
Reduces Recombination Yes (recA1)
Shipping Condition Dry Ice
T1 Phage - Resistant (tonA) No
Species E. coli (K12)
Format Tube
Product Line MAX Efficiency
Quantity 5 x 200 μL
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Frequently Asked Questions (FAQs)

I am trying to clone an insert that is supposedly pretty toxic. I used DH5? and TOP10 cells for the transformation and got no colonies on the plate. Do you have any suggestions for me?

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

- After transforming TOP10 or DH5? cells, incubate at 25-30°C instead of 37°C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
- Try using TOP10F' cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
- Try using Stbl2 cells for the transformation.
How do you recommend that I prepare my DNA for successful electroporation of E. coli?

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.
You offer competent cells in Subcloning Efficiency, Library Efficiency and MAX Efficiency. How do these differ?

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:

Subcloning Efficiency cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA
How can I clone a gene that has direct repeats and propagate it without altering the repeat sequences?

The first thing you can do is to lower the growth temperature of your E. coli cells when propagating your plasmid containing the unstable gene. Slowing the growth of any cell strain at 30C, 25C or even lower can help to stabilize the replication of the plasmids they contain.

If your sequence is still unstable despite low-temperature growth, there are also specific bacterial strains available that can further help to stabilize repeated sequences during propagation. Invitrogen Stbl2 and Stbl4 competent cells are both designed to improve stability when cloning retroviral or direct repeat sequences.

In a series of experiments, Stbl2 was compared directly to several other strains also known for increasing stability of retroviral and tandem repeat inserts. An article in the Focus Journal (Issue 16.3, p. 78) contains data from two such experiments – the full article can be found on the Thermo Fisher Scientific website. A brief summary of the data is included below:

Stability of clones containing SIV retroviral sequences:
Stbl2 @ 30°C - 100%; Stbl2 @ 37°C - 100%; HB101 @ 30°C - 100%; HB101 @ 37°C - 100%; SURE @ 30°C - 72%; SURE @ 37°C - 0%

Stability of clones containing 100 repeats of a 32-bp sequence:
Stbl2 @ 30°C - 89%; Stbl2 @ 37°C - 73%; HB101 @ 30°C - 15%; HB101 @ 37°C - 0%; SURE @ 30°C - 53%; SURE @ 37°C - 0%

Results from a separate experiment on stability of a tandem repeat of four R67 dihydrofolate reductase genes in Stbl2 vs. SURE cells can be found in Focus 19.2, p. 24 on the Thermo Fisher Scientific website.
Do any Invitrogen competent cells contain DMSO in the freezing medium?

Yes, several of our competent cells products are frozen with DMSO. The presence of DMSO (dimethylsulfoxide) will generally be indicated in the MSDS files if you have a question about a particular product, but here is a list of commonly used products that are known to have DMSO in the freezing buffer:

One Shot OmniMAX 2 T1 Phage Resistant Cells, Cat. No. C8540-03

One Shot INV?F' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot MAX Efficiency DH5?-T1 Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency DH5?-T1 Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency DH5? Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency DH5? Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency DH5? F'IQ Cells, Cat. No. 18288-019

MAX Efficiency Stbl2Chemically Competent Cells, Cat. No. 10268-019
Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.
How can unstable or toxic DNA inserts be maintained in bacteria?

There are a few steps you can take to improve stability of clones with difficult-to-maintain inserts. Supplement the medium with extra nutrients (e.g., add 20-30 mM glucose to Terrific Broth) or try a vector that has a reduced copy number (e.g., pBR322). Some clones can exhibit a high degree of deletions; this is usually a result of the clones having long terminal repeat (LTR) sequences or regions with high secondary structure. To overcome this problem, the cells can be grown at 30°C or ambient temperature (in LB or in a nutrient rich broth like Terrific Broth). Do not to let the cells reach late stationary phase in liquid culture. Alternatively, transform into cells that maintain unstable sequences such as Stbl2, Stbl3, or Stbl4 cells.
Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

For Research Use Only. Not for use in diagnostic procedures.